Automated parallel capillary electrophoresis system with hydrodynamic sample injection

ABSTRACT

An automated capillary zone electrophoretic system is disclosed. The system employs a capillary cartridge having a plurality of capillary tubes. The cartridge has a first array of capillary ends projecting from one side of a plate. The first array of capillary ends are spaced apart in substantially the same manner as the wells of a microtitre tray of standard size. This allows one to simultaneously perform capillary electrophoresis on samples present in each of the wells of the tray. The system includes a stacked, dual carrousel arrangement to eliminate cross-contamination resulting from reuse of the same buffer tray on consecutive executions from electrophoresis. The system also has a container connected to the detection end of the capillaries. The container is provided with valving which facilitate cleaning the capillaries, loading buffer into the capillaries, introducing samples to be electrophoresced into the capillaries, and performing capillary zone electrophoresis on the thus introduced samples.

RELATED APPLICATIONS

This application is a Continuation under 35 U.S.C. § 120 of U.S. patentapplication Ser. No. 09/388,125, filed Aug. 31, 1999, now U.S. Pat. No.6,352,633.

TECHNICAL FIELD

This invention relates to an automated apparatus for performingmultiplexed Capillary Electrophoresis. It is especially useful in anautomated Capillary Zone Electrophoresis (CZE) system for loadingsamples into a plurality of capillaries from wells of commerciallyavailable, microtitre trays of standard size.

BACKGROUND

The contents of commonly-owned U.S. patent application Ser. No.09/105,988, which issued as U.S. Pat. No. 6,027,627 and also waspublished as WO 99/00664 are incorporated by reference to the extentnecessary to understand the present invention. This reference disclosesan automated apparatus for capillary electrophoresis.

FIG. 1 illustrates a prior art automated electrophoretic apparatusdiscussed in the above-referenced patent application for capillaryelectrophoresis. The apparatus includes a light source 452, aprocessor/controller 404, a dual carrousel arrangement having an uppercarrousel 601 and a lower carrousel 602 which are aligned and spacedapart along a common axis and operated by a rotor 604, a DC motor 605having a movable member 603 to move a tray 214 place on one of thecarrousels along a common axis toward or away from an array of capillaryends belonging to a capillary cartridge 300, a detector 408 fordetecting, at a window region 130 of the capillaries, the fluorescenceemitted by samples migrating along the capillaries, and a computermonitor 406 to view the results of the migration. An electrophoreticmedium, such as a gel, can be introduced into the capillaries via aconduit 606 in preparation for an electrophoretic run.

FIG. 2 illustrates a prior art plumbing system in accordance with theabove-identified reference, for performing capillary electrophoresisusing the device of FIG. 1. In particular, FIG. 2 shows the integrationof a gel syringe 804 and an HPLC wash solvent system 807 into thesolvent/gel delivery module. A solvent manifold 850 connects threeinlets from the feeder tubes 806 of the solvent containers 801, 802, 803to an outlet. Feeder tubes 806 from the solvent containers 801, 802, 803are connected to the inlets of the solvent manifold 850 by tubing 860.The controller 404 pictured in FIG. 1 controls the solvent manifold 850to select solvent from one of the three solvent containers 801, 802,803. The inlet of the HPLC pump 807 is connected to the outlet of thesolvent manifold 850 by tubing 861 and the outlet of the HPLC pump 807is connected to an inlet of a valve manifold 851 by tubing 862.

The valve manifold 851 connects two inlets and an outlet. One inlet ofthe valve manifold 851 is connected to the gel syringe 804 by tubing 863and the other inlet of the valve manifold 851 is connected to the outletof the HPLC pump 807. The outlet of the valve manifold 851 is connectedto the solvent/gel input port 606 by tubing 864. The controller 404pictured in FIG. 11 causes the valve manifold 851 to select either theinlet connected to the gel syringe 804 or the inlet connected to theHPLC pump 807. In this manner, gel and solvents are delivered to thecapillary cartridge 909 in preparation for capillary gel electrophoresisof samples in microtitre tray 852.

In the preferred embodiment, the tubing connecting the feeder tubes 806of the solvent containers 801, 802, 803 to the inlets of the solventmanifold 850 is standard teflon tubing with a diameter of ⅛ inches. Thetubing 861 connecting the outlet of the solvent manifold 850 to theinlet of the HPLC pump 807 is PEEK tubing with a diameter of 1/16inches. The tubing 861 connecting the outlet of the solvent manifold 850to the inlet of the HPLC pump 807, the tubing 862 connecting the outletof the HPLC pump 807 to an inlet of the valve manifold 851, the tubing863 connecting the gel syringe 804 to an inlet of the valve manifold 851and the tubing 864 connecting the outlet of the valve manifold 851 tothe solvent/gel input port are PEEK tubing with a diameter of 1/16inches.

FIG. 3 illustrates a preferred embodiment of capillary cartridge 1180 inaccordance with the above-identified application. In this embodiment,the capillary tubes run from their first ends 1188 disposed in anelectrode/capillary array 1181. The capillary tubes then run insidemultilumen tubing 1183. The multilumen tubing is taught in detail inU.S. patent application Ser. No. 08/866,308, which is incorporated byreference herein. The multilumen tubing 1183 is held firmly in place bytubing holders 1185. The capillary tubes, without the protection themultilumen tubing, pass through an optical detection region 1187. Beyondthe optical detection region 1187, the capillary tubes have a commontermination and are bundled together and cemented into a high pressureT-shaped fitting 1182 made from electrically conductive material, which,during electrophoresis, is connected to electrical ground.

The tubing holders 1185 and the T-fitting 1182 are fixed to a cartridgebase 1186. The cartridge base 1186 is made from polycarbonate plasticfor its dielectric characteristic. The base 1186 in turn is removablyattached to a shuttle 1179 which includes a set of rail couplings 1184protruding from its bottom. These rail couplings 1184 are arranged sothat they fit on to a railing system (not shown in FIG. 18) of theapparatus in FIG. 1. The railing system allows the shuttle 1184 to movebetween an in position and out position. The base 1186 is detached fromthe shuttle 1179 so that the cartridge 1180 is disposed (or cleaned) anda new (or cleaned) capillary cartridge is attached when the shuttle 1179is in its out position. The combination of the railing system and theshuttle 1179 allows the newly attached capillary cartridge to berepeatedly located at the same position as that of the disposedcapillary cartridge in relation to a camera and a laser (not shown inFIG. 3) when the shuttle 1179 is in its in position. In a preferredembodiment, the shuttle 1179 extends the length of the base 1186 with anopening to accommodate the electrode/capillary array 1181; the shuttle1179 is attached to the base 1186 by a plurality of removable fasteners1178.

The prior art plumbing system of FIG. 2 and T-fitting of FIG. 3 are bestsuited for capillary gel electrophoresis. In capillary gelelectrophoresis, the gel is fairly viscous, on the order of 50,000centi-poise. This requires a system which can create pressure sufficientto load gel into the capillaries in preparation for a capillaryelectrophoresis run, and sufficient to expel the gel from thecapillaries during reconditioning.

In contrast to the gels that are used in capillary gel electrophoresis,buffers are used to load the capillaries in capillary zoneelectrophoresis (CZE). These buffers have a viscosity on the order ofthat of water, i.e., about 1 centi-poise. While the low viscosity ofbuffers has the advantage of not needing high pressure to load andunload the electrophoretic medium, CZE with buffers does have thedisadvantage of capillary siphoning. Capillary siphoning ischaracterized by the buffer solution at one end of the capillaries beingcompletely drawn into the capillaries, thereby depleting the buffer atthat one end. Like siphoning of any tubing, this problem occurs when thetwo ends of the capillaries terminate at different heights. The obvioussolution to this problem is to ensure that opposite ends of thecapillaries are maintained at the same level. This, however, is lessthan an ideal solution.

SUMMARY OF THE INVENTION

The present invention is directed to an automated parallel capillaryzone electrophoresis (CZE) system. The CZE system of the presentinvention is realized by modifying the prior art capillary gelelectrophoresis (CGE) system of the above-reference prior art. Moreparticularly, the present invention is principally realized by modifyingthe plumbing at the ends of the capillaries towards which samples in thecapillaries migrate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a side view of a prior art automated capillary electrophoresissystem suitable for capillary gel electrophoresis;

FIG. 2 illustrates a prior art plumbing system for the electrophoresissystem of FIG. 1;

FIG. 3 is a side view of a prior art capillary cartridge for use withthe electrophoresis system of FIGS. 1 and 2; and

FIG. 4 a shows a preferred embodiment of the present invention forperforming capillary zone electrophoresis;

FIG. 4 b shows a sequence of valve settings for the embodiment of FIG. 4a;

FIG. 5 shows a second embodiment of a system in accordance with thepresent invention;

FIGS. 6 a & 6 b show two versions of a third embodiment of a system inaccordance with the present invention;

FIG. 7 shows intensity images comprising fluorescence data fromexperimental samples in 96 capillaries simultaneously migrating; and

FIGS. 8 a, 8 b & 8 c shows intensity plots for experimental samplesmigrating in three of the 96 capillaries.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The contents of commonly-owned, aforementioned U.S. patent applicationSer. No. 09/105,988, which issued as U.S. Pat. No. 6,027,627 and alsowas published as WO 99/00664, are incorporated by reference to theextent necessary to understand the present invention.

FIG. 4 a shows a buffer cell 100 connected to a capillary cartridge 102via a pressure fitting 104 not unlike that shown in FIG. 3. Indeed,capillary cartridge 102 is similar in structure to the capillarycartridge 1180 of FIG. 3, except that capillary cartridge 102 does notinclude the T-fitting 1182. In the present invention, the buffer cell100 and its associated hardware shown in FIG. 4 a replace the prior artT-fitting 1182 of FIG. 3 and some of the prior art plumbing system seenin FIG. 2.

The buffer cell 100 has a interior cavity 106 which is which preferablyis sealed from the exterior, except for openings discussed below. In thepreferred embodiment, the cell is formed from an acrylic plastic, whichis an electrical insulating material. Inner walls of the cell are shapedand sized to provide an interior cavity 106 into which a buffer or otherliquid 112 may be introduced. In the preferred embodiment, the containerhas a capacity of about 100 ml, by volume.

A high voltage electrode 110 connected to a power supply (not shown) isin contact with the liquid 112 in the cell 100. for the purpose ofapplying a predetermined potential to the liquid in the container, andthereby also to the first, cell ends 107 of the capillaries which are incommunication with the liquid 112. During CZE, the high voltageelectrode 110 is held at ground, while a non-zero voltage is applied tothe second, sample ends 108 of the capillaries, with the polarity of thevoltage being determined by the charge-type of the samples beingseparated. The magnitude of the applied voltage is on the order of 10-15kV, not unlike that used in capillary gel electrophoresis.

A plurality of conduits communicate with the cavity 106 viacorresponding valves. In the preferred embodiment, the valves aresolenoid valves or the like, which can be controlled by computer, muchas discussed in the above-identified U.S. application Ser. No.09/105,888. In FIG. 4 a, each of the five conduits connected to the cell100, whether it is an inlet or an outlet, or serves as both, is shown tohave a separate valve. It is understood, however, that one or more ofthese valves may be internal to equipment connected to the correspondingconduit, rather than being a discrete valve.

Drain outlet 114 and drain valve 116 allow a liquid in the cavity 106 toexit the cell 100 into a waste container (not shown). Air conduit 118and gas (air) release valve 120 provide a path from the interior of thecavity 106 to the atmosphere when air valve release 120 is open. Pumpinlet 122 and pump valve 124 provide a path for buffers, solvents andother liquids in containers, such as those indicated by 801, 802 and803, to enter the cell 100 via one or manifolds 850, under assistance ofan HPLC pump 807, or the like. Pressure conduit 126 and pressure valve128 connect a syringe 130 or other pressure applicator to the cavity 106at a point above the level of liquid 112 therein. Finally, overflowoutlet 132 and overflow valve 134 cooperate to provide a passage fromthe interior of the cavity 106 to a waste container, so as to ensurethat the cell 100 does not overfill. While the various valves 116, 120,124, 128 and 134 are shown to be distinct devices, it should be kept inmind that one or more of these valve may be an integral part of anotherdevice. For instance, pump valve 124 may be integrally formed as part ofHPLC pump 807, and pressure valve 128 may be replaced by preciselycontrolling the syringe's piston 136 by a stepper motor, or the like,under the direction of a controller.

FIG. 4 a depicts the valve positions for performing steps associatedwith preparing and conducting electrophoresis on the samples in thecapillary tubes of the capillary cartridge 102.

When the cell 100 is to be drained, the pressure valve 128 and the pumpvalve 124 are closed, and the drain valve 116 and at least one, if notboth, of the air valve 120 and the overflow valve 134 are opened. Thisallows the liquid in the cell to drain via drain conduit 114.

Once the cell 100 has been completely drained, it may be partiallyfilled with a liquid. For this, the drain valve 116 and the pressurevalve 128 are closed, and the pump valve 124 and at least one, if notboth, of the air valve 120 and overflow valve 134 open. The pump 807 isthen operated to introduce a selected one of the liquids in containers801, 802, 803 into the cell 100. Because the pump introduces liquid intothe reservoir and, because at least one of the air valve 120 and theoverflow valve 132 is open, the liquid is not forced into thecapillaries. However, the pump is controlled to turn off when the liquidreaches a predetermined level within the cell.

When the capillaries are to be cleaned, a cleaning solution, or thelike, present in one or more of the containers 801, 802, 803, is forcedinto the cell 100, into the cell ends 107 of the capillary tubes, andout the sample ends 108 of the capillary tubes. For this, only the pumpvalve 124 is open while all the other valves are closed. Under suchconditions, when the HPLC pump 807 operates, it forces liquid into thecell 106, increasing the pressure therein. The increased pressure forcesthe cleaning solution into the cell ends 107, through the capillarytubes and out the sample ends 108. Once cleaning solution has beenforced through, the pump valve may be closed, and the cell 100 drained,as discussed above.

After cleaning, the cell can be filled with buffer to a predeterminedlevel by selecting the appropriate container 801, 802, 803 with themanifold 850, and operating the pump 807 with the drain valve 116 andthe pressure valve 128 closed, and the pump valve 124 and at least one,if not both, of the air valve 120 and overflow valve 134 open. Thepredetermine level of buffer should exceed the level of the bundle ofcapillary cell ends 107.

Once the level of the buffer has exceeded the level of the capillarycell ends 107, buffer may be loaded into the capillaries. For this, theonly the pump valve 124 is left open, and all other valves are closed.The buffer enters the capillary cell ends 107, thereby forcing anymaterial within the capillary tubes out the capillary sample ends 108into a waste container (not shown), and loading the capillary tubes withbuffer. At this point, the cell 100 is filled with buffer to just belowthe level of the overflow conduit 132, yet above the level of thecapillary cell ends. In the preferred embodiment, the overflow conduit132 is at about the 60% fill level and so the cell 100, having acapacity of 100 ml, contains approximately 60 ml of buffer.

It should be evident that filling the capillaries with buffer is similarto the procedure for cleaning the capillaries, except that buffer,rather than a cleaning solvent, is used. As discussed above, this iscontrolled by operating the manifold 850 connected to the containers 80,802 and 803 holding buffers, cleaning solutions and other liquids. Itshould be noted, however, that buffer itself can be used to clean thecapillaries

To introduce a sample into the sample ends 108 of the capillaries, thesample ends 108 are first dipped into wells of a microtitre tray ofstandard size, such as those having a rectangular array of 8 rows of 12wells, or those having 16 rows of 24 wells. The wells contain thesamples to be electrophoresced.

The samples can be introduced into the sample ends 108 of thecapillaries in one of two ways. One way is electro-kinetic injectionwherein a voltage differential is applied between the sample ends andthe cell ends of the capillaries so as to cause a portion of the sampleto enter the sample ends. During electro-kinetic injection, the airvalve 120 is kept open keep the reservoir 100 at atmospheric pressure,equilibrated with the cell ends 107 of the capillary. By applying a highvoltage differential, the electro-osmotic flow causes sample enter thecapillary sample ends 108. Once the sample has been introduced into thesample ends from the wells of the microtitre tray, the sample tray isreplaced a buffer tray and electrophoretic separation can take place inthe capillaries under high voltage.

A second way in which to load samples into the sample ends 108 of thecapillaries is by hydrodynamic injection. First, air valve 120 is openedand all other valves are closed to equilibrate both ends of thecapillaries with atmospheric pressure. After equilibration, the airvalve 120 is also closed, and so no valves are left open. At this point,the plunger 136 of the syringe 130 is pulled back by a predeterminedvolume. This causes the air above the liquid level in the cell to expandinto a slightly greater volume and thereby create a vacuum, or negativepressure. At this point, the pressure valve 128 is opened, therebyapplying this negative pressure to the surface of the buffer 112 in thecell 100. Due to the negative pressure, a small amount of sample (orother substance in each of the wells of the microtitre tray) is suckedin at each of the capillary sample ends. However, because air expands tofill the volume, there is a slight time lag between opening the pressurevalve 128 and the uptake of sample. After the sample is allowed to enterdue to the negative pressure for a predetermined period of time,typically on the order of a few seconds, the air valve 120 is opened,thereby stopping the injection process. Experiments have shown thathydrodynamic injection produces more reproducible results, and more evensample injection into the capillaries. This is because the volume intowhich the air expands does not immediately cause an instantaneous,corresponding intake of sample at the capillary sample ends, when thepressure valve 126 is opened. Instead, a fairly even uptake into each ofthe capillary sample results.

The pulling volume of the syringe controls the degree of negativepressure or vacuum. In the preferred embodiment, the plunger is pulledback by an amount sufficient to displace about 2 ml. In a 100 mlcontainer having 60 ml of buffer therein, there is about 40 ml of air.When the plunger is pulled back by 2 ml, a negative pressure (relativeto atmospheric) of 2.0 ml/40.0 ml=0.05 atm (or about 0.7 psi) isgenerated. Assuming a syringe precision of 0.1 ml and a container volumeof 100 ml, the precision of the negative pressure can be controlled toabout 0.001 atm.

Once the sample has been introduced into the capillary sample ends, thesample tray is preferably replaced by a buffer tray in preparation forelectrophoresis. Replacing the sample trays with buffer trays helpsensure than excess sample is not taken into the capillary tubes, andalso ensures that both ends of the capillary tubes are inserted intobuffer. Using a device in accordance with the present invention,electrophoresis can take place in either a static mode, or a dynamicmode.

In the static mode, the pump 807 is not operational and only the airvalve 120, or the overflow valve 134, or both, are open, with theremaining valves closed. Under these conditions, the buffer in the cell112 is substantially stagnant during electrophoresis.

In the dynamic mode, the pressure valve 128 is closed, and all othervalves are open, and the pump is operational, with buffer continuouslybeing pumped into the cell through the pump inlet 122 and exiting thecell via drain outlet 114. This ensures that fresh buffer bathes thecapillary cell ends during electrophoresis while older buffer drainsfrom the cell. Samples which have completed migrating from the sampleend all the way to the cell end are also drained through drain outlet114 and drain valve 116. At the same time, since air conduit 118 and airvalve 120 are open, the atmospheric pressure at both ends of thecapillaries is equalized, thereby counteracting the siphoning effect,especially when the capillary ends are at the same height.

The dynamic mode, in which there is continuous flushing of the cell 100,provides several advantages. First, continuously providing fresh buffersolution to the capillary cell ends removes charge depletion duringelectrophoresis. Charge depletion happens when anion and cation layersbuild up around the electrode, thereby resulting in a voltage dropbetween these layers which, in turn, reduces the voltage drop across thecapillary tubes for separation. Flowing buffer helps retard theformation of such layers so that sample separation is more reproduciblefrom run to run.

A second advantage to constant flushing is that it assists in removingfluids and contaminants introduced into the cell by electro-osmotic flow(EOF) during electrophoresis. EOF is a continuous pumping process whichbrings small amounts of sample-laden buffer into the cell. This cancause a change in buffer conductivity during electrophoresis. Constantflushing helps mitigate the problem of a solute-imbalance. Sensors andfeedback control systems connected to the pump and to the pump and drainvalves can ensure that the liquid level in the cell is maintained at apredetermined level.

A third advantage to continuous flushing is that it reduces the timespent cleaning the capillary tubes between runs. Because fresh buffer isconstantly being introduced into the cell in the dynamic mode, one needspend as much time rinsing out the cell, upon conclusion of each run.

A fourth advantage to continuous flushing is that it removes air bubbleswhich otherwise collect around the capillary cell ends 107 duringelectrophoresis. Such removal is believed to be brought about by thebuffer flowing past this area.

In one example of continuous flushing using capillaries with an innerdiameter of 50 μm, a voltage differential of 10 kV across the capillaryends and borate buffer at a pH of 10.5, EOF speed is about 12 cm/min.This causes the liquid volume of the cell to increase at the rate ofabout 53 μl/min. If a drain is provided, the buffer must be replenished,as needed. In the preferred embodiment, only about 1 ml/min of freshbuffer is introduced into the cell while the drain valve is openedduring electrophoresis.

Despite the above-stated advantages, it should be kept in mind thatcontinuous flushing, though preferable, is not an absolute requirementin the present invention. Indeed, the primary requirements for carryingout CZE in accordance with the present invention are that a cell beprovided, the cell having a liquid therein with the capillary cell endsterminating in said liquid, and that some mechanism be provided forcreating a vacuum, or suction effect, at the capillary cell ends so asto draw samples into capillary sample ends.

FIG. 5 presents another embodiment in accordance with the presentinvention. In the embodiment of FIG. 5, a sealed, or at least sealable,cell 100 partially filled with a liquid 112 is provided. The capillarycell ends 107 terminate in this liquid 112. An air syringe 130 and anHPLC pump 807 are also provided. When the syringe plunger 136 is pulledin the direction shown by the arrow A1, sample is introduced into thecapillary sample ends 108, as depicted by arrow A2. As discussed abovewith reference to FIG. 4 a, conduits for drain, air release and overflowmay also be provided. To clean the cell in this embodiment, one simplyrestrains the syringe plunger and runs the pump to flush out the liquidin the cell and in the capillary tubes via the capillary second ends.

FIG. 6 a presents yet another embodiment in accordance with the presentinvention. In this embodiment, which is similar to embodiment of FIG. 5,the entire cell and the syringe are filled with liquid and no air (orother gas) is used. Unlike air, liquid is incompressible, and so thereis neither a time delay nor a variation in volume, between pulling thesyringe plunger and the introduction of samples into the capillarysample ends. This means that the syringe must be much more preciselycontrolled in the embodiment of FIG. 6 a than in the embodiment of FIG.5. For this, a micro-syringes operated by high-precision stepper motors,or the like, is used to ensure that only a small quantity of sample,about 0.1 μl or so, per capillary, is introduced into each of thecapillary second ends. To clean the cell and the capillary tubes in theembodiment of FIG. 6 a, one may either push on the syringe plunger orrun the pump; either one forces buffer into the cell and out through thecapillary sample ends.

FIG. 6 a presents still another embodiment in accordance with thepresent invention. In this embodiment, the syringe is replaced by anarrow-diameter drain outlet 140 controlled by a valve 142 situated at avertical position lower than that of the capillary sample ends 108. Inthis embodiment, gravity is used to cause a negative pressure. With thepump off, when the valve 142 is opened, liquid drains through theconduit 140 as indicated by arrow A3. This siphons liquid into thecapillary sample ends, as indicated by arrow A4.

In the embodiments of FIGS. 5, 6 a and 6 b, discrete valves between thepump and the cell are not shown; it is understood, however, that suchvalves may be integral with the pump. Similarly, no such valves areshown between the syringe and the cell. As explained above, the syringeplunger may be restrained and controlled by a motor so as to exertsufficient force in the appropriate direction, as dictated by amicroprocessor or other controller. Also, with regard to the embodimentsof FIGS. 6 a and 6 b, it is noted that since only a very minute quantityof liquid is introduced from the capillary tubes into the cell, there isno appreciable increase in pressure within the cell, which issubstantially able to accommodate the added amount.

EXPERIMENTAL EXAMPLE

In an experimental set-up, capillary zone electrophoresis was carriedout simultaneously in 96 capillaries using a device substantiallyarranged as shown in FIG. 4 a. About 60 ml of buffer was introduced intoa 100 ml cell. The buffer used was a 10 mM borate solution in de-ionizedwater, adjusted to a pH 10.5 with NaOH. The viscosity of the buffer wasalmost the same as that of water.

Ninety-six capillaries, each having a length of about 50 cm, and ID of50 μm and an 150 OD μm, available from Polymicro Technology of Phoenix,Ariz. were used. A window region was burned into each capillary using ahot wire at a point approximately 10 cm from one end of the capillaries,thereby providing an effective migration distance of about 40 cm fromthe sample end to the window region at which sample detection would takeplace. The capillaries were arranged substantially parallel to oneanother in a ribbon-like arrangement. More specifically, for most oftheir length from the sample ends to the window, the capillaries werespaced apart from one another by about 150 μm and, at the window region,were spaced apart by about 300 μm. Beyond the window region, the cellends of the 96 capillaries were bound together as a bundle with TorrSeal, available from Varian Vacuum Products of Lexington, Mass. Thisbundle was connected to the cell shown in FIG. 4 a with a Swagelockfitting, with the capillaries being in communication with the buffer.Meanwhile, the sample ends of the capillaries formed a two-dimensionalarray with a spacing corresponding to that of the wells of an 8×12microtitre tray of standard size.

A 3 μl sample was introduced into each of the wells of an 8×12microtitre tray. The sample comprised a protein cluster separated fromamong a multitude of such clusters in a protein mixture extracted frombacteria. The proteins were labeled with fluorescein dye, which has itsabsorption maximum at 495 nm. The sample ends of the capillaries wereinserted into corresponding wells of the microtitre tray, in contactwith the sample therein. Samples in each of the 96 wells were thenhydrodynamically injected into the sample ends of the capillaries. Thiswas performed by creating a vacuum by pulling on the syringe plunger todisplace a 3 ml volume with all valves closed, and holding the plungerin place. At this point, the pressure valve was opened, thereby causinga negative pressure at the air-buffer interface on the surface of thebuffer in the cell. The pressure valve was opened for about 20 seconds,permitting sufficient time for sample to be sucked into each of thecapillary sample ends. At this point, the air valve was opened toalleviate the negative pressure and stop further hydrodynamic injectionof sample.

Next, the microtitre tray containing samples was replaced with amicrotitre tray containing buffer, in preparation for electrophoresis. Avoltage differential of 10 kV was applied for about 10 minutes acrossthe 50 cm-long capillaries, thereby providing an electric field of 200v/cm and causing the samples to migrate under electro-osmotic flow,along with the buffer. An all-line Argon-ion laser, available fromSpectra-Physics of Mountain View, Calif., and having an emissions peaknot far from 495 nm, was used to illuminate the capillariessubstantially at right angles thereto at the window region duringelectrophoresis. A CCD camera, available from PixelView of Beaverton,Oreg., was used to detect the fluorescence of the samples as they passedthrough the window region of the capillaries. The camera was set upsubstantially as disclosed in co-owned allowed U.S. application Ser. No.09/084,236, also published as WO 99/32877.

FIG. 7 shows the fluorescence intensities at 530±8 nm, as a function oftime, of the samples in the 96 capillaries, In FIG. 7, the abscissa(x-axis) represents the capillary number while the ordinate (y-axis)represents time. The darker the spot, the higher the intensity.

FIGS. 8 a, 8 b and 8 c show plots of relative intensities for edge andcenter capillaries (capillary nos. 1, 48 and 96) in the array, as afunction of time. In FIG. 8, the abscissa (x-axis) represents time,while the ordinate (y-axis) represents the intensity. As seen in FIG. 8,the intensity contours are substantially the same, exhibiting similarpeaks from each capillary, albeit at slightly different migration timesfor each capillary.

As seen in this experimental example, CZE can be used to separateproteins in a buffer having a predetermined pH. For example, CZE can beused for human growth hormone separation, Ca++ binding proteinseparation, and recombinant human erythroprotein protein separation,among others. The separation mechanism in CZE is based on the ratio ofthe net charge to the size of the proteins. The net charge can be ofeither polarity, depending on the buffer pH and the protein's structure.Electro-osmotic flow of the buffer in the capillaries sweeps neutralmolecules, as well as charged proteins, toward the detection window. Thebuffer preferably has a viscosity about the same as that of water.

The present invention may also be used in other capillaryelectrophoresis settings in which the separation media has lowviscosity, on the order of 1-150, and more preferably on the order of1-50, centipoise. At these viscosities, the separation media can bepumped into the capillaries under pressure without damage to thecapillaries or other components of the system, and the samples injectedhydrodynamically. A number of these other approaches and applicationsare now discussed.

Sodium Dodecyl Sulfate(SDS)-type Capillary Gel(CGE)/NGE(Non-Gel)Electrophoresis. In this approach, the proteins are bound withthe surfactant SDS to form negatively charged aggregates. Apolymer-based sieving matrix, such as polyethylene oxide(PEO),preferably kept at a low pH to extend the lifetime of the capillaries,is used as the separation medium. Applications for this include peptidemapping, molecular weight estimation, protein quantization and proteinstability analysis. In some cases, CGE with a low-viscosity separationmedia, such as polyvinylpyrrolidone (PVP), which has a viscosity of 1-25centipoise when in a weight percentage of 0.1-5%, can be used for DNAseparation, as reported in Gao & Yeung, Anal. Chem., 1998, v. 70, pp.1382-1388.

Capillary Iso-Electric Focusing (CIEF), in which the proteins areseparated according to their unique iso-electric points in a separationmedium having a viscosity similar to that of water, may also beperformed using the device and method of the present invention.

Affinity Capillary Electrophoresis (ACE) in which proteins are separatedon the basis of specific bonding to other molecules in a separationmedium having a viscosity of about 5-50 centipoise may also be performedusing the device and method of the present invention.

Micellular Electrokinetic Capillary Chromotography (MEKC),in whichcompounds are separated based on their hydro-phobicity in a separationmedium having a viscosity of about 5-50 centipoise may also be performedusing the device and method of the present invention. Such an approachwould be espcially useful in separating non-charged species.

Capillary Isotachphoresis (CITP), which is used for in-capillary proteinpre-concentration, immediately preceding CZE, may be performed using thedevice and method of the present invention.

While the above invention has been described with reference to certainpreferred embodiments, examples and suggested applications, it should bekept in mind that the scope of the present invention is not limited tothese. One skilled in the art may find variations of these preferredembodiments which, nevertheless, fall within the spirit of the presentinvention, whose scope is defined by the claims set forth below.

1. A capillary zone electrophoresis subsystem configured to cooperatewith a light source and a light detector to detect migrating samples,said subsystem comprising: a fluid container; a plurality of capillarytubes, each capillary tube having a first end and a second end, thefirst ends being arranged to receive samples thereinto and the secondends terminating at a first level in the fluid container; a power supplyconfigured to apply a voltage across the first and second ends of thecapillary tubes; a pump connected to said fluid container via a pumpconduit, said pump configured to introduce a liquid into the containerwhen said pump conduit is open and said pump is operating; a vacuumdevice connected to said fluid container via a vacuum conduit enteringsaid fluid container at a second level higher than said first level,said vacuum device configured to cause a negative pressure in saidcontainer, when said pump conduit is closed and said container issealed; a gas release valve connected to said container and configuredto vent a gas in the container when said gas release valve is opened;and a drain valve connected to said container and configured to drain aliquid in said container, when said drain valve is open.
 2. Thecapillary zone electrophoresis subsystem of claim 1, further comprising:an overflow conduit connected to said container, said overflow conduitconfigured to release liquid held within the container, when a height ofsaid liquid within the container exceeds a predetermined level.
 3. Thecapillary zone electrophoresis subsystem of claim 2, further comprising:an overflow valve positioned in said overflow conduit, said overflowvalve having at least a first, open position, and a second, closedposition.
 4. The capillary zone electrophoresis subsystem of claim 1,wherein said vacuum device is a syringe.
 5. The capillary zoneelectrophoresis subsystem of claim 1, wherein said capillary first endsare arranged in a two-dimensional array having a spacing correspondingto that of wells of a microtitre tray, said system further comprising: apositioning apparatus comprising an upper and a lower carrousel carryingmicrotitre trays, said positioning apparatus arranged to position one ofsaid microtitre trays such that said two dimensional array of capillaryfirst ends is inserted into corresponding wells of said microtitre tray.6. The capillary zone electrophoresis subsystem of claim 1, wherein thevacuum device and the pump are separate devices.
 7. A capillary zoneelectrophoresis subsystem configured to cooperate with a light sourceand a light detector to detect migrating samples, said subsystemcomprising: a fluid container; a plurality of capillary tubes, eachcapillary tube having a first end and a second end, the first ends beingarranged to receive samples thereinto and the second ends terminating ata first level in the fluid container; a power supply configured to applya voltage across the first and second ends of the capillary tubes; apump connected to said fluid container via a pump conduit, said pumpconfigured to introduce a liquid into the container when said pumpconduit is open and said pump is operating; and a vacuum deviceconnected to said fluid container via a vacuum conduit entering saidfluid container at a second level higher than said first level, saidvacuum device configured to cause a negative pressure in said container,when said pump conduit is closed and said container is sealed, whereinthe vacuum device and the pump are separate devices.
 8. The capillaryzone electrophoresis subsystem of claim 7, further comprising: a gasrelease valve connected to said container and configured to vent a gasin the container when said gas release valve is opened.
 9. The capillaryzone electrophoresis subsystem of claim 8, further comprising: a drainvalve connected to said container and configured to drain a liquid insaid container, when said drain valve is open.
 10. The capillary zoneelectrophoresis subsystem of claim 9, further comprising: an overflowconduit connected to said container, said overflow conduit configured torelease liquid held within the container, when a height of said liquidwithin the container exceeds a predetermined level.
 11. The capillaryzone electrophoresis subsystem of claim 10, further comprising: anoverflow valve positioned in said overflow conduit, said overflow valvehaving at least a first, open position, and a second, closed position.12. The capillary zone electrophoresis subsystem of claim 7, whereinsaid capillary first ends are arranged in a two-dimensional array havinga spacing corresponding to that of wells of a microtitre tray, saidsystem further comprising: a positioning apparatus comprising an upperand a lower carrousel carrying microtitre trays, said positioningapparatus arranged to position one of said microtitre trays such thatsaid two dimensional array of capillary first ends is inserted intocorresponding wells of said microtitre tray.
 13. The capillary zoneelectrophoresis subsystem of claim 7, wherein said vacuum device is asyringe.
 14. The capillary zone electrophoresis subsystem of claim 7,wherein said vacuum device is a drain conduit provided with a valvemember positioned at a height below a height of said capillary firstends, whereby a gravity flow of liquid through said drain conduit bygravity causes a negative pressure in said container, thereby siphoningsamples into each of said plurality of capillary tube first ends.
 15. Amethod for capillary zone electrophoresis on a plurality of samples,said method comprising: providing a fluid container; providing aplurality of capillary tubes, each capillary tube having a first end anda second end, the first ends being arranged to receive samples thereintoand the second ends terminating at a first level in the fluid container;introducing a first liquid into said fluid container to a height atleast as high as said first level; applying a negative pressure to saidcapillary second ends to hydrodynamically introduce a sample to beelectrophoresced into each of said capillary first ends, wherein thestep of applying a negative pressure is performed by a device distinctfrom that used for the step of introducing a first liquid; and applyinga voltage differential between said capillary first ends and saidcapillary second ends to cause said samples to migrate towards saidcapillary second ends through electro-osmotic flow.
 16. The methodaccording to claim 15, wherein the step of applying a negative pressurecomprises: withdrawing air in said liquid container at a point above alevel of said liquid while said container is sealed, thereby causingsamples to enter said capillary first ends.
 17. The method according toclaim 16, wherein the step of withdrawing air comprises: retracting aplunger of a syringe connected to said liquid container.
 18. The methodaccording to claim 15, wherein said step of applying a negative pressurecomprises: draining a portion of a liquid in said liquid container, suchthat samples are siphoned.
 19. The method according to claim 15, furthercomprising: flowing a liquid past said capillary second ends whileapplying said voltage differential.
 20. The method according to claim19, wherein the step of applying a negative pressure compriseswithdrawing air in said liquid container at a point above a level ofsaid liquid while said container is sealed, thereby causing samples toenter said capillary first ends.
 21. The method according to claim 20,wherein the step of withdrawing air comprises: retracting a plunger of asyringe connected to said liquid container.
 22. The method according toclaim 19, wherein the step of applying a negative pressure comprisesdraining a portion of a liquid in said liquid container, such thatsamples are siphoned.